Influenza is an enveloped, single-stranded, negative-sense RNA virus that causes serious respiratory ailments throughout the world. It is the only member of the Orthomyxoviridae family and has been subgrouped into three types, A, B and C.
Influenza virions consist of an internal ribonucleoprotein core containing the single-stranded RNA genome and an outer lipoprotein envelope lined inside by a matrix (hereinafter “M1”) protein. The segmented genome of influenza A consists of eight molecules of linear, negative polarity, single-stranded RNA sequences that encode ten polypeptides. Segment 1 is 2341 nucleotides in length and encodes PB2, a 759 amino acid polypeptide which is one of the three proteins which comprise the RNA-dependent RNA polymerase complex.
The remaining two polymerase proteins, PB1, a 757 amino acid polypeptide, and PA, a 716 amino acid polypeptide, are encoded by a 2341 nucleotide sequence and a 2233 nucleotide sequence (segments 2 and 3), respectively. Segment 4 of the genome consists of a 1778 nucleotide sequence encoding a 566 amino acid hemagglutin (HA) surface glycoprotein which projects from the lipoprotein envelope and mediates attachment to and entry into cells. Segment consists of 1565 nucleotides encoding a 498 amino acid nucleoprotein (NP) protein that forms the nucleocapsid. Segment 6 consists of a 1413 nucleotide sequence encoding a 454 amino acid neuramimidase (NA) envelope glycoprotein. Segment 7 consists of a 1027 nucleotide sequence encoding a 252 amino acid M1 protein, and a 96 amino acid M2 protein, which is translated from a spliced variant of the M RNA. Segment 8 consists of a 890 nucleotide sequence encoding two nonstructural proteins, NS1 and NS2, composed of 230 and 121 amino acids respectively, whose function is not well defined. NS2 is translated from a spliced variant of the NS RNA.
The segmented genome of influenza B consists of eight molecules of linear, negative polarity, single-stranded RNA sequences that encode eleven polypeptides. Segment 2 is 2396 nucleotides in length and encodes PB2, a 770 amino acid polypeptide which is one of the three RNA-dependent RNA polymerase proteins. The remaining two influenza B polymerase proteins, PB1, a 752 amino acid polypeptide, and PA, a 725 amino acid polypeptide, are encoded by a 2386 nucleotide sequence and a 2304 nucleotide sequence (segments 1 and 3), respectively. Segment 4 of the genome consists of a 1882 nucleotide sequence encoding a 584 amino acid HA surface glycoprotein which projects from the lipoprotein envelope and mediates attachment to cells and membrane fusion. Segment 5 consists of 1839-1841 nucleotides encoding a 560 amino acid NP protein that forms the nucleocapsid. Segment 6 consists of a 1454 nucleotide sequence encoding a 466 amino acid NA envelope glycoprotein and a 100 amino acid NB protein, a nonstructural protein whose function is unknown. Segment 7 consists of a 1191 nucleotide sequence encoding a 248 amino acid M1 protein and a 195 amino acid BM2 protein which is translated from a separate reading frame. Segment 8 consists of a 1096 nucleotide sequence encoding nonstructural proteins NS1 and NS2, composed of 281 and 122 amino acids respectively, whose functions are not well defined. NS2 is translated from a spliced variant of the NS RNA.
The segmented genome of influenza C consists of seven molecules of linear, negative polarity, single-stranded RNA sequences that encode eight polypeptides. Segment 1 is 2365 nucleotides in length and encodes PB2, a 774 amino acid polypeptide which is one of the three RNA-dependent RNA polymerase proteins. The remaining two polymerase proteins, PB1, a 754 amino acid polypeptide, and PA, a 709 amino acid polypeptide, are encoded by a 2363 nucleotide sequence and a 2183 nucleotide sequence (segments 2 and 3), respectively.
Segment 4 of the genome consists of a 2074 nucleotide sequence encoding a 655 amino acid hemagglutinin-esterase surface glycoprotein which projects from the lipoprotein envelope and mediates attachment to cells, fusion, and has receptor-destroying activities. Segment 5 consists of a 1809 nucleotide sequence encoding a 565 amino acid NP protein that forms the nucleocapsid. Segment 6 consists of a 1180 nucleotide sequence encoding a 374 amino acid matrix (M) protein. Segment 7 consists of a 934 nucleotide sequence encoding a 286 amino acid NS1 protein, and a 122 amino acid NS2 protein, which is translated from a spliced variant of the NS RNA.
To infect a cell influenza HA protein adsorbs to sialyloligosaccharide molecules in cell membrane glycoproteins and glycolipids. Following endocytosis of the virion, a conformational change in the HA molecule occurs within the cellular endosome that facilitates membrane fusion and triggers uncoating. The nucleocapsid migrates to the nucleus where viral mRNA is transcribed as the essential initial event in infection. Transcription and replication of influenza RNA take place in the nucleus of infected cells and assembly into virions occurs by budding out of or through the plasma membrane. The viruses can reassort genes during mixed infections.
Replication of influenza virus RNAs is dependent on four viral gene products: PB1, PB2, PA, and NP. The three polymerase proteins, PB1, PB2, and PA, form a trimolecular complex in the nuclei of infected cells. Each protein has its own nuclear localization signal. See Akkina, J. Virol 61:2217-24 (1987), Mukaigawa, J Virol 65:245-253 (1991) and Nieto, J Gen Virol 75: 29-36 (1994). Some specific functions have been ascribed to the individual polypeptides. PB1 appears to be primarily involved in the enzymatic polymerization process, i.e. the elongation step. It shares regions of amino acid homology with other RNA-dependent RNA polymerase proteins. The precise function of PA is unknown. The PB2 protein binds to the 5′-terminal cap structure present on host cell mRNAs; the mRNAs are then cleaved, producing a capped 9 to 15-mer oligoribonucleotide which serves as a primer for transcription of influenza mRNAs. The PB2 amino acid sequence contains a region of limited homology with the cellular cap-binding protein, eIF4E. See de la Luna, Virus Res 13:143-56 (1989). While PB2 is not absolutely required for replication of viral RNA, mRNAs transcribed from viral template in cells expressing only PB1, PA, and NP are uncapped and thus cannot be translated. See Nakagawa, J Virol 69:728-33 (1995). Transcripts terminate at sites 15-22 bases from the ends of their templates, where oligo(U) sequences act as signals for the template-independent addition of poly(A) tracts. At a later stage of infection, instead of making mRNAs, the polymerase proteins PB1, PB2 and PA are used to make new viral RNA genomes. The polymerase complex first transcribes cRNA, which then serves as template for production of more vRNA. The plus-stranded cRNA copies differ from the plus-stranded mRNA transcripts by lacking capped and methylated 5′-termini. Also, they are not truncated or polyadenylated at the 3′ termini. Thus, the cRNAs are coterminal with their negative strand templates and contain all the genetic information in each genomic segment in the complementary form. The negative strand genomes (vRNAs) and antigenomes (cRNAs) are always encapsidated by viral nucleocapsid proteins; the only unencapsidated RNA species are virus mRNAs. Nucleocapsid assembly appears to take place in the nucleus. The virus matures by budding from the apical surface of the cell incorporating the M1 protein on the cytoplasmic side or inner surface of the budding envelope. The HA and NA glycoproteins are incorporated into the lipid envelope. In permissive cells, HA is post-translationally cleaved, but the two resulting chains remain associated by disulfide bonds.
Efforts to produce immunogenic compositions against influenza have taken two paths. Inactive vaccines, which cannot replicate in the host, can be either chemically inactivated whole virus or viral subunit proteins. Both inactivated and subunit virus vaccines are available for influenza. These vaccines contain the HA and NA surface proteins as antigens which give rise to the immune response upon administration to the host. For reasons which are incompletely understood, subunit vaccines have exhibited an efficacy of only 60% to 80% against influenza disease. Inactivated whole virus vaccines are administered intramuscularly and primarily stimulate a systemic immune response, whereas live attenuated vaccines also stimulate local mucosal immunity. The latter form of immunity is more effective since it is present in the upper respiratory tract where the virus is first encountered. Also, inactivated vaccines typically have reduced ability to induce cytotoxic T cell responses, and can sometimes cause delayed hypersensitivity reactions. Guillain-Barre syndrome has been associated with the inactivated influenza A “swine flu” vaccine. See, Schonberger, Ann Neurol 9(supp):31-38(1981).
Live attenuated viruses can be employed in immunogenic compositions and are typically successful at inducing the required protective response in the host. Live attenuated influenza viruses are capable of limited replication in the host, thus stimulating a protective immune response, but without causing disease. Previously, such mutants have been generated by multiple passage through an unnatural host such as embryonated chicken eggs, by successive passage through an unnatural host at increasingly lower temperatures, or by random mutagenesis via chemical methods and selection of conditional mutants. These methods can result in the loss of pathogenicity while retaining immunogenicity. However, the identity of the genetic mutations generated as described above are unknown a priori and when the mutant “master donor” virus is selected as a vaccine candidate. If such mutations are limited to one or two nucleotide changes, the virus composition could ultimately “revert” or back mutate in the host and thus regain its original pathogenic phenotype. However, one of these methods, successive passage at increasingly lower temperatures, has given rise to a virus (the “cold-adapted” strain derived from A/Ann Arbor/6/60) with multiple mutations that has been shown to be genetically stable. See Murphy, If Dis In Clin Practice 2: 174-181 (1993). In making such vaccine compositions, the HA and NA RNA sequences of the attenuated master donor virus are replaced with HA and NA RNA sequences from circulating influenza strains. Such viruses are termed reassortant viruses.
Temperature sensitive (ts) mutants of influenza, generated by chemical mutagenesis or identified by screening for spontaneous mutants have been described. Such mutants are unable to replicate in the lower respiratory tract of infected animals and often replicate in the upper respiratory tract to a lower level than wild-type virus. One of these mutants, ts 1A2, was shown to have many of the desired characteristics of a live attenuated influenza vaccine. See Murphy and Chanock, Genetic Variation Among Influenza Viruses, pps 601-615, Nayak, D. ed, Academic Press, NY (1981) and Murphy, Phil Trans R Soc Lon B 288:401-15(1980). The ts1A2 strain was found to contain temperature sensitive lesions in both PB1 and PB2, and exhibited the desired level of attenuation but was genetically unstable and reverted to a virulent state after replication in a seronegative young vaccinee. See Murphy, Ann NY Acad Sci 354:172-82 (1980) and Tolpin, Infection and Immunity 36:645-50 (1982).
A panel of temperature sensitive mutants of the A/Udorn/307/72 whose ts lesions were mapped to the PB2 gene has been described. Sequence analysis led to the discovery of mutations at amino acid positions 65, 100, 112, 171, 298, 310, 386, 391 and 556 of PB2. Similarly, the PB2 gene of the ts1A2 virus was found to have a mutation at amino acid position 65×. See Lawson, Virology 191:506-10 (1992). The cold-adapted strain of A/AA/6/60 is also temperature sensitive and sequence analysis has suggested that one of the mutations which may be partly responsible for the ts phenotype is an asparagine to serine change at amino acid position 265 of PB2. See Cox, Virology 167:554-67 (1988), Herlocher, Proc Natl Acad Sci 90:6032-36 (1993) and Snyder, J Virol 62:488-95 (1988). Additionally, PB2 ts mutants of A/WSN/33 and of A/FPV/Rostock/34 are known. The mutation in the PB2 gene sequence, presumably responsible for the ts phenotype, was localized to amino acid 417 in the case of A/WSN/33 and to amino acid 512 in the case of A/FPV/Rostock/34. See McCauley, Virus Res 17:191-98 (1990) and Yamanaka, Arch Virol 114:65-73 (1990). Collectively, these studies suggest many potential sites in the PB2 protein where mutations may be introduced in order to generate a ts virus.
An alternate method of creating a live attenuated virus is by employing the techniques of “reverse genetics”. See Enami, Proc Natl Acad Sci 87:380205(1990), Enami and Palese, J Virol 65:2711-13(1991) and Luytjes, Cell 59:1107-13 (1989). In this process, modified vRNA-like transcripts are transcribed in vitro from cDNA constructs in the presence of purified NP, PB1, PB2, and PA proteins. The resulting synthetic RNP is then transfected into cells previously infected with influenza virus, usually a helper virus which has a conditional growth defect, such as host range restriction or temperature sensitivity, which allows the subsequent selection of transfectant viruses. For example, host-range helper viruses have been successfully used to rescue synthetic NA and PB2 genes. See Enmi, supra, and Subbarao, J Virol 67:7223-7228 (1993). Antibody selection can also be used to select transfectants in the case of the HA and NA genes. Using antibody selection techniques, the surface HA glycoprotein gene has been transfected and rescued into influenza A virus. See, Horimoto and Kawaoka, J Virol 68:3120-3128 (1994) and Li, J Virol 66:399-404(1992). The HA gene has also been transfected and rescued into influenza B virus. See, Barclay and Palese, J Virol 69:1275-1279 (1995). The M gene (see, Yasuda, J Virol 68:8141-8146 (1994)), and the NP gene (see Li, Virus Res, in press), has also been rescued using the techniques of reverse genetics.
Given the possibility of using reverse genetics to engineer specific mutations into the genome of influenza, it should be possible to create a ts strain with mutations that are less likely to revert and thus exhibit the desired property of genetic stability. This may be accomplished by introducing new codons which would require more than one nucleotide within the codon to mutate in order to encode the wild-type amino acid, by mutating sites which are less likely to be suppressed extragenically, or by introducing multiple, independently-acting mutations in one or more genes. Since only four of the amino acid changes described above can be engineered such that more than one base change is required in order to revert to a codon that encodes the wild-type amino acid, the identification of additional sites for the introduction of ts mutations would be highly desirable.
“Clustered charged-to-alanine mutagenesis” is a technique whereby charged amino acids are mutagenized to the uncharged amino acid alanine so as to maintain the overall structure or stability of the protein while modifying its bioactivity. It has been used to create mutants of the human growth hormone receptor protein (see Bass, Proc Natl Acad Sci 88:44984502 (1991)), the Saccharomyces cervisiae actin protein (see Wertman, Genetics 132:337-50 (1995)), the poliovirus 3D polymerase protein (see Diamond and Kirkegaard, J Virol 68:863-76 (1994)), the vaccinia virus G2R protein (see Hassett and Condit, Proc Natl Acad Sci 91:45544559 (1994)), and human immunodeficiency virus type 1 integrase protein (see Wiskerchen, J Virol 69:597-601 (1995)). In each of the foregoing cases, a “charged cluster” was defined as a sequence of five contiguous amino acids, at least two of which are charged.